Regulation of MUC5AC expression by NAD(P)H:quinone oxidoreductase 1

Abstract

Neutrophil elastase (NE), a potent neutrophil inflammatory mediator, increases MUC5AC mucin gene expression through undefined pathways involving reactive oxygen species. To determine the source of NE-generated reactive oxygen species, we used pharmacologic inhibitors of oxidoreductases to test whether they blocked NE-regulated MUC5AC mRNA expression. We found that dicumarol, an inhibitor of the NADP(H):quinone oxidoreductase 1 (NQO1), inhibited MUC5AC mRNA expression in A549 lung adenocarcinoma cells and primary normal human bronchial epithelial cells. We further tested the role of NQO1 in mediating NE-induced MUC5AC expression by inhibiting NQO1 expression using short interfering RNA (siRNA). Transfection with siRNA specific for NQO1 suppressed NQO1 expression and significantly abrogated MUC5AC mRNA expression. NE treatment caused lipid peroxidation in A549 cells; this effect was inhibited by pretreatment with dicumarol, suggesting that NQO1 also regulates oxidant stress in A549 cells after NE exposure. NE exposure increased NQO1 protein and activity levels; NQO1 expression and activity were limited to the cytosol and did not translocate to the plasma membrane. Our results indicate that NQO1 has an important role as a key mediator of NE-regulated oxidant stress and MUC5AC mucin gene expression.

Figure 1

Dicumarol blocks the NE-induced increase in MUC5AC mRNA expression in A549 cells. A549 cells were treated with dicumarol (4-30 μM) 1h prior to and then during NE (100 nM, 6h) or control vehicle treatment. RNA was isolated using Trizol reagent as per the manufacturer's instructions, or by the GTC/CsCl method, and used for quantitative real-time RT-PCR (A) or for Northern analyses (10 μg) (B). MUC5AC mRNA expression determined by real-time RT-PCR was normalized to 18s ribosomal RNA and expressed relative to control treated cells using the ΔΔC_t method (n=4-8, 3 separate experiments, *, significantly different from control, p=0.0002: #, significantly different from NE alone, p=0.014,A). A representative northern analysis for MUC5AC mRNA expression is shown [C, Control; D, 30μM dicumarol ; NE, 100nM neutrophil elastase; ND, neutrophil elastase and dicumarol] (B). The filter was stripped and reprobed for 28s rRNA. Molecular markers (9.5 & 4.4 kb) are shown with arrows. The graph summarizes the northern densitometry data of MUC5AC mRNA levels normalized to 28s rRNA levels and expressed as a percentage of control (C). Data are presented as mean ±SEM (n=6, 3 separate experiments). The dashed line at 100% represents control levels. *, significantly different from control (P < 0.05). #, significantly different from NE alone (P < 0.05).

Figure 2

Dicumarol blocks the NE-induced increase in MUC5AC mRNA expression in NHBE cells. NHBE cells grown on Transwell clear chambers (10 days at ALI) were starved in media supplemented with only two factors, BSA and RA, for 24h and treated with dicumarol (4 or 10 μM) or vehicle 1h prior to and then during NE (500 nM, 1h) or control vehicle treatment. Total RNA was collected by Trizol. MUC5AC mRNA expression was evaluated by quantitative real-time RT-PCR. The relative MUC5AC mRNA level was normalized to 18S rRNA and expressed as percent of control treated cells. Data are presented as mean ± SEM (3 separate experiments with total n of 4-6). The dashed line at 100% represents control levels in the absence of dicumarol and NE.*, significantly different from control treated cells (P=0.002). #, significantly different from NE alone (P = 0.041).

Figure 3

Knockdown of NQO1 expression by NQO1 siRNA. A549 (70% confluent) were transfected with siRNA for NQO1 (siNQO1, 20 nM), or control siRNA (siControl, 20 nM). 24 h post-transfection, total RNA was collected by Trizol. NQO1 mRNA expression was evaluated by quantitative real-time RT-PCR. The relative NQO1 mRNA level was normalized to 18S rRNA and expressed as percent of control siRNA (siControl) transfected cells. Data are presented as mean ± SEM (n=9, 3 separate experiments). *, significantly different from siControl transfected cells (P<0.0001) (A). Another set of cells with the same treatment were used to collect cell lysate and NQO1 protein expression was evaluated by Western blot (B). The graph summarizes the densitometry data of NQO1 protein level normalized to β-actin and expressed as a percentage of siControl (C). Data are presented as mean ±SEM (n=4, 2 separate experiments). *, significantly different from siControl (P < 0.03).

Figure 4

NQO1-knockdown by siRNA transfection abrogates the NE-induced upregulation of MUC5AC mRNA expression. A549 cells (70% confluent) were transfected with siRNA for NQO1 (siNQO1, 20 nM), or control siRNA (siControl, 20 nM). 22h post-transfection, cells were treated with NE (100nM) or control vehicle for 6h and total RNA was collected by Trizol. MUC5AC mRNA expression was evaluated by quantitative real-time RT-PCR and normalized to 18s rRNA expression. The relative MUC5AC mRNA levels from NE-treated cells were expressed as a percentage of their corresponding control-treated cells. Data are presented as mean ± SEM (n=9, 3 separate experiments). The dashed line at 100% represents control levels. *, significantly different from siControl transfected cells (P<0.04).

Figure 5

Dicumarol inhibited NE-induced lipid peroxidation in A549 cells. A549 cells were preincubated with dicumarol (30μM, 1h) or 0.1M NaOH and then treated with NE (100nM) or control vehicle for 2h. Cell lysate was collected and evaluated for lipid peroxidation by HPLC. Results are expressed as percent of control. D, dicumarol; NE, neutrophil elastase; NE+D, neutrophil elastase plus dicumarol. Data are presented as mean ± SEM (n=10, 2 separate experiments). The dashed line at 100% represents control levels. *, significantly different from control, P<0.006. #, significantly different from NE (P=0.021).

Figure 6

NE increased NQO1 protein and activity level in A549 whole cell lysates. Whole cell lysates of A549 cells following treatment with NE (100nM, 1h) or control vehicle (C) were evaluated for NQO1 protein levels by western blot (A). The graph summarizes the densitometry data of NQO1 protein level normalized to Ponceau S staining. Results are expressed as a percent of control, mean ± SEM (n=10, 4 separate experiments). *, significantly different from control (P= 0.0002) (B). NQO1 activity in whole cell lysate was evaluated using a spectrophotometric assay measuring the reduction of DCPIP at 600 nm. Relative NQO1 activity was expressed as a percent of control, mean ± SEM (n=10, 4 separate experiments). *, significantly different from control (P = 0.0022) (C).

Figure 7

NE increased NQO1 protein and activity levels in NHBE whole cell lysates. Whole cell lysates of NHBE following treatment with NE (500 nM, 1 h) or control vehicle (Ctrl) were evaluated for NQO1 protein levels by western analysis (A). The graph summarizes the densitometry data of NQO1 protein levels normalized to β-actin. Results are expressed as a percent of control (C), mean ± SEM (n=9-12, 4 separate experiments). *, significantly different from control, (p=0.0006) (B). NQO1 activity in the whole cell lysate was evaluated using a spectrophotometric assay measuring the reduction of DCPIP at 600 nm in the presence or absence of dicumarol. Relative NQO1 activity was expressed as a percent of control, mean ±SEM (n=5, 2 separate experiments). *, significantly different from control (P = 0.016) (C).

Figure 8

NE increased NQO1 protein and activity level in the cytosolic fraction, not in the membrane fraction. Cytosolic and membrane fractions of A549 cells treated with NE (100nM, 1h) or control vehicle (C) were evaluated for NQO1 protein level by western blot (A). E-cadherin was used as a positive control for the plasma membrane fraction (B). The graph summarizes the densitometry data of NQO1 protein level in the cytosol normalized to Ponceau S staining. Results are expressed as a percent of control, mean ±SEM (n=6, 4 separate experiments). *, significantly different from control (P= 0.01) (C). NQO1 activity in the cytosolic fraction was evaluated using a spectrophotometric assay measuring the reduction of DCPIP at 600 nm. Relative NQO1 activity was expressed as a percent of control, mean ±SEM (n=6, 4 separate experiments). *, significantly different from control (P = 0.002)(D).